The world's most widely grown fruit crop, the grape (Vitis sp.), is cultivated on all continents except Antarctica. However, major grape production centers are in European countries (including Italy, Spain, and France), which constitute about 70% of the world grape production (Mullins et al., Biology of the Grapevine, Cambridge, U.K.:University Press (1992)). The United States, with 300,000 hectares of grapevines, is the eighth largest grape grower in the world. Although grapes have many uses, a major portion of grape production (˜80%) is used for wine production. Unlike cereal crops, most of the world's vineyards are planted with traditional grapevine cultivars, which have been perpetuated for centuries by vegetative propagation. Several important grapevine virus and virus-like diseases, such as grapevine leafroll, corky bark, and Rupestris stem pitting, are transmitted and spread through the use of infected vegetatively propagated materials. Thus, propagation of certified, virus-free materials is one of the most important disease control measures. Traditional breeding for disease resistance is difficult due to the highly heterozygous nature and outcrossing behavior of grapevines, and due to polygenic patterns of inheritance. Moreover, introduction of a new cultivar may be prohibited by custom or law. Recent biotechnology developments have made possible the introduction of special traits, such as disease resistance, into an established cultivar without altering its horticultural characteristics.
Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses, and nematodes can infect grapes, and the resultant diseases can cause substantial losses in production (Pearson et al., Compendium of Grape Diseases, American Phytopathological Society Press (1988)). Among these, viral diseases constitute a major hindrance to profitable growing of grapevines. About 34 viruses have been isolated and characterized from grapevines. The major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus, other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy (1993)).
Of the major virus diseases, the grapevine leafroll complex is the most widely distributed throughout the world. According to Goheen (“Grape Leafroll, ” in Frazier et al., eds., Virus Diseases of Small Fruits and Grapevines (A Handbook), University of California, Division of Agricultural Sciences, Berkeley, Calif., USA, pp. 209-212 (1970) (“Goheen (1970)”), grapevine leafroll-like disease was described as early as the 1850s in German and French literature. However, the vital nature of the disease was first demonstrated by Scheu (Scheu, “Die Rollkrankheit des Rebstockes (Leafroll of grapevine),” D. D. Weinbau 14:222-358 (1935) (“Scheu (1935)”)). In 1946, Harmon and Snyder (Harmon et al., “Investigations on the Occurrence, Transmission, Spread and Effect of ‘White’ Fruit Colour in the Emperor Grape, ” Proc. Am. Soc. Hort. Sci. 74:190-194 (1946)) determined the viral nature of White Emperor disease in California. It was later proven by Goheen et al. (Goheen et al., “Leafroll (White Emperor Disease) of Grapes in California, Phytopathology, 48:51-54(1958), (“Goheen (1958)”)) that both leafroll and “White Emperor” diseases were the same, and only the name leafroll was retained.
Leafroll is a serious viral disease of grapes and occurs wherever grapes are grown. This wide distribution of the disease has come about through the propagation of diseased vines. It affects almost all cultivated and rootstock varieties of Vitis. Although the disease is not lethal, it causes yield losses and reduction of sugar content. Scheu estimated in 1936 that 80 per cent of all grapevines planted in Germany were infected (Scheu, Mein Winzerbuch, Berlin:Reichsnahrstand-Verlags (1936). In many California wine grape vineyards, the incidence of leafroll (based on a survey of field symptoms conducted in 1959) agrees with Scheu's initial observation in German vineyards (Goheen et al., “Studies of Grape Leafroll in California,” Amer. J. Enol. Vitic., 10:78-84 (1959)). The current situation on leafroll disease does not seem to be any better (Goheen, “Diseases Caused by Viruses and Viruslike Agents,” The American Phytopathological Society, St. Paul, Minn.:APS press, 1:47-54 (1988) (“Goheen (1988)”). Goheen also estimated that the disease causes an annual loss of about 5-20 per cent of the total grape production (Goheen (1970) and Goheen (1988)). The amount of sugar in individual berries of infected vines is only about ½ to ⅔ that of berries from noninfected vines (Goheen (1958)).
Symptoms of leafroll disease vary considerably depending upon the cultivar, environment, and time of the year. On red or dark-colored fruit varieties, the typical downward rolling and interveinal reddening of basal, mature leaves is the most prevalent in autumn; but not in spring or early summer. On light-colored fruit varieties however, symptoms are less conspicuous, usually downward rolling accompanied by interveinal chlorosis. Moreover, many infected rootstock cultivars do not develop symptoms. In these cases, the disease is usually diagnosed with a woody indicator indexing assay using Vitis vivifera cv. Carbernet Franc (Goheen (1988)).
Ever since Scheu demonstrated that leafroll was graft transmissible, a virus etiology has been suspected (Scheu (1935)). Several virus particle types have been isolated from leafroll diseased vines. These include potyvirus-like (Tanne et al., “Purification and Characterization of a Virus Associated with the Grapevine Leafroll Disease,” Phytopathology, 67:442-447 (1977)), isometric virus-like (Castellano et al., “Virus-like Particles and Ultrastructural Modifications in the Phloem of Leafroll-affected Grapevines,” Vitis, 2:23-39 (1983) (Castellano (1983)”) and Namba et al., A Small Spherical Virus Associated with the Ajinashika Disease of Koshu Grapevine, Ann. Phytopathol. Soc. Japan, 45:-70-73 (1979)), and closterovirus-like (Namba, “Grapevine Leafroll Virus, a Possible Member of Closteroviruses, Ann. Phytopathol, Soc. Japan, 45.497-502 (1979)) particles. In recent years, however, long flexuous closteroviruses ranging from 1,400 to 2,200 nm have been most consistently associated with leafroll disease (FIG. 1) (Castellano (1983), Faoro et al., “Association of a Possible Closterovirus with Grapevine Leafroll in Northern Italy,” Riv. Patol. Veg. Ser IV, 17:183-189 (1981), Gugerli et al., “L'enroulement de la vigne; mise en évidence de particules virales et développement d'une méthode immuno-enzymatique pour le diagnostic rapide (Grapevine Leafroll: Presence of Virus Particles and Development of an Immuno-enzyme method for Diagnosis and Detection),” Rev. Suisse Viticult. Arboricult, Hort., 16:299-304 (1984) (“Gugerli (1984)”), Hu et al., “Characterization of Closterovirus-like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathol., 128:1-14(1990) (“Hu (1990)”), Milne et al., “Closterovirus-like Particles of Two Types Associated with Diseased Grapevines,” Phytopathol, Z., 110:360-368(1984), Zee et al., “Cytopathology of Leafroll-diseased Grapevines and the Purification and Serology of Associated Closterovirus like Particles,” Phytopathology, 77:1427-1434(1987) (“Zee (1987)”), and Zimmerman et al., “Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,” J. Phyopathol., 130:205-218 (1990) (“Zimmermann (1990)”)). These closteroviruses are referred to as grapevine leafroll associated viruses (“GLRaV”). At least six serologically distinct types of GLRaV's (GLRaV-1 to -6) have been detected from leafroll diseased vines (Table 1) (Boscia et al., “Nomenclature of Grapevine Leafroll-associated Putative Closteroviruses, Vitis, 34:171-175 (1995) (“Boscia (1995)”) and (Martelli, “Leafroll,” pp. 37-44 in Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, Rome Italy, (1993) (“Martelli I”)). The first five of these were confirmed in the 10th Meeting of the International Council for the Study of Virus and Virus Diseases of the Grapevine (“ICVG”) (Volos, Greece, 1990).
TABLE 1CoatParticle lengthprotein MrType(nm)(×103)ReferenceGLRaV-11,400-2,20039Gugerli (1984)GLRaV-21,400-1,80026Gugerli (1984)Zimmermann (1990)GLRaV-31,400-2,20043Zee (1987)GLRaV-41,400-2,20036Hu (1990)GLRaV-51,400-2,20036Zimmermann (1990)GLRaV-61,400-2,20036Gugerli (1993)Through the use of monoclonal antibodies, however, the original GLRaV II described in Gugerli (1984) has been shown to be an apparent mixture of at least two components, IIa and IIb (Gugerli et al., “Grapevine Leafroll Associated Virus II Analyzed by Monoclonal Antibodies,” 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 23-24 (1993) (“Gugerli (1993)”)). Recent investigation with comparative serological assays (Boscia (1995)) demonstrated that the IIb component of cv. Chasselas 8/22 is the same as the GLRaV-2 isolate from France (Zimmermann (1990)) which also include the isolates of grapevine corky bark associated closteroviruses from Italy (GCBaV-BA) (Boscia (1995)) and from the United States (GCBaV-NY) (Namba et al., “Purification and Properties of Closterovirus-like Particles Associated with Grapevine Corky Bark Disease,” Phytopathology, 81:964-970 (1991) (“Namba (1991)”)). The IIa component of cv. Chasselas 8/22 was given the provisional name of grapevine leafroll associated virus 6 (GLRaV-6). Furthermore, the antiserum to the CA-5 isolate of GLRaV-2 produced by Boscia et al. (Boscia et al., “Characterization of Grape Leafroll Associated Closterovirus (GLRaV) Serotype II and Comparison with GLRaV Serotype III, “Phytopathology, 80:117 (1990)) was show to contain antibodies to both GLRaV-2 and GLRaV-1, with a prevalence of the latter (Boscia (1995)).
Virions of GLRaV-2 are flexuous, filamentous particles about 1,400-1,800 nm in length (Gugerli et al., “L'enroulement de la Vigne: Mise en Evidence de Particles Virales et Development d'une Methode Immuno-enzymatique Pour le Diagnostic Rapide (Grapevine Leafroll: Presence of Virus Particles and Development of an Immuno-enzyme Method for Diagnosis and Detection),” Rev. Suisse Viticult, Arboricult, Horticult. 16:299-304 (1984)). A double-stranded RNA (dsRNA) of about 15 kb was consistently isolated from GLRaV-2 infected tissues (Goszczynski et al., Detection of Two Strains of Grapevine Leafroll-Associated Virus 2,” Vitis 35:133-35 (1996)). The coat protein of GLRaV-2 is ca 22˜26 kDa (Zimmermann et al., Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,” J. Phytopathology 130:205-18 (1990); Gugerli and Ramel, Extended abstracts: “Grapevine Leafroll Associated Virus II Analyzed by Monoclonal Antibodies,” 11th ICVG at Montreux, Switzerland, Gugerli, ed., Federal Agricultural Research Station of Changins, CH-1260 Nyon, Switzerland, p 23-24 (1993); Boscia et al., “Nomenclature of Grapevine Leafroll-Associated Putative Closteroviruses,” Vitis 34-171-75 (1995)), which is considerably smaller than other GLRaVs (35˜43 kDa) (Zee et al., “Cytopathology of Leafroll-Diseased Grapevines and the Purification and Serology of Associated Closterovirus Like Particles,” Phytopathology 77:1427-34 (1987); Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. of Phytopathology 128:1-14(1990); Ling et al., “The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3Cloning, Nucleotide Sequencing and Expression in Transgenic Plants,” Arch. of Virology 142:1101-16(1997)). Although GLRaV-2 has been classified as a member of the genus Closterovirus based on particle morphology and cytopathology (Martelli, Circular of ICTV-Plant Virus Subcommittee Study Group on Closterolike Viruses” (1996)), its molecular and biochemical properties are not well characterized.
In the closterovirus group, several viruses have recently been sequences. The partial or complete genome sequences of beet yellows virus (BYV) (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72:15-24 (1991), Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198:311-24 (1994)), beet yellow stunt virus (BYSV) (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996)), citrus tristeza virus (CTV) (Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus Tristeza Closterovirus Genome,” Virology 199:35-46 (1994); Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208:511-20 (1995)), lettuce infectious yellows virus (LIYV) (Klaassen et al., “Partial Characterization of the Lettuce Infectious Yellows Virus Genomic RNAs, Identification of the Coat Protein Gene and Comparison of its Amino Acid Sequence With Those of Other Filamentous RNA Plant Viruses,” J. General Virology 75:1525-33 (1994); Klaassen et al., “Genome Structure and Phylogenetic Analysis of Lettuce Infectious Yellows Virus, a Whitefly-Transmitted, Bipartite Closterovirus,” Virology 208:99-110 (1995)), little cherry virus (LChV) (Keim and Jelkmann, “Genome Analysis of the 3′-Terminal Part of the Little Cherry Disease Associated dsRNA Reveals a Monopartite Clostero-Like Virus,” Arch. Virology 141:1437-51 (1996); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78:2067-71 (1997)), and GLRaV-3 (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3-Genome Reveals a Typical Monopartite Closterovirus,” J. Gen. Virology 79(5):1289-1301 (1998)) revealed several common features of the closteroviruses, including the presence of HSP70 chaperone heat shock protein and a duplicate of the coat protein gene (Agranovsky “Principles of Molecular Organization, Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv. in Virus Res. 47:119-218 (1996); Dolja et al. “Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology 32:261-85 (1994); Boyko et al., “Coat Protein Gene Duplication in a Filamentous RNA Virus of Plants,” Proc. Nat. Acad. Sci. USA 89:9156-60 (1992)). Characterization of the genome organization of GLRaVs would provide molecular information on the serologically distinct closteroviruses that cause similar leafroll symptoms in grapevine.
Several shorter closteroviruses (particle length 800 nm long) have also been isolated from grapevines. One of these, called grapevine virus A (“GVA”) has also been found associated, though inconsistently, with the leafroll disease (Agran et al., “Occurrence of Grapevine Virus A (GVA) and Other Closteroviruses in Tunisian Grapevines Affected by Leafroll Disease,” Vitis, 29:43-48 (1990), Conti, et al., “Closterovirus Associated with Leafroll and Stem Pitting in Grapevine,” Phytopathol. Mediterr., 24:110-113 (1985), and Conti et al., “A Closterovirus from a Stem-pitting-diseased Grapevine,” Phytopathology, 70:394-399 (1980)). The etiology of GVA is not really known; however, it appears to be more consistently associated with rugose wood serau lato (Rosciglione at al., “Maladies de l'enroulement et du bois strié de la vigne: analyse microscopique et sérologique Leafroll and Stem Pitting of Grapevine: Microscopical and Serological Analysis),” Rev. Suisse Vitic Arboric. Hortic., 18:207-211 (1986) (“Rosciglione (1986)”), and Zimmermann (1990)). Moreover, another short closterovirus (800 nm long) named grapevine virus B (“GVB”) has been isolated and characterized from corky bark-affected vines (Boscia et al., “Properties of a Filamentous Virus Isolated from Grapevines Affected by Corky Bark,” Arch. Virol., 130:109-120(1993) and Namba(1991)).
As suggested by Martelli I, leafroll symptoms may be induced by more than one virus or they may be simply a general plant physiological response to invasion by an array of phloem-inhabiting viruses. Evidence accumulated in the last 15 years strongly favors the idea that grapevine leafroll is induced by one (or a complex) of long closteroviruses (particle length 1,400 to 2,200 nm).
Grapevine leafroll is transmitted primarily by contaminated scions and rootstocks. However, under field conditions, several species of mealybugs have been shown to be the vector of leafroll (Engelbrecht et al., “Transmission of Grapevine Leafroll Disease and Associated Closteroviruses by the Vine Mealybug Planococcus-ficus,” Phytophylactica, 22:341-346 (1990), Rosciglione, et al., “Transmission of Grapevine Leafroll Disease and an Associated Closterovirus to Healthy Grapevine by the Mealybug Planococcus ficus,” (Abstract), Phytoparasitica, 17:63-63 (1989), and Tanne, “Evidence for the Transmission by Mealybugs to Healthy Grapevines of a Closter-like Particle Associated with Grapevine Leafroll Disease,” Phytoparasitica, 16:288 (1988)). Natural spread of leafroll by insect vectors is rapid in various parts of the world. In New Zealand, observations of three vineyards showed that the number of infected vines nearly doubled in a single year (Jordan et al., “Spread of Grapevine Leafroll and its Associated Virus in New Zealand Vineyards,” 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 113-114 (1993)). One vineyard became 90% infected 5 years after GLRaV-3 was first observed. Prevalence of leafroll worldwide may increase as chemical control of mealybugs becomes more difficult due to the unavailability of effective insecticides.
In view of the serious risk grapevine leafroll virus poses to vineyards and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. The present invention is directed to overcoming this deficiency in the art.